CN109385411A - A kind of beta-Mannosidase and its application - Google Patents
A kind of beta-Mannosidase and its application Download PDFInfo
- Publication number
- CN109385411A CN109385411A CN201710665651.6A CN201710665651A CN109385411A CN 109385411 A CN109385411 A CN 109385411A CN 201710665651 A CN201710665651 A CN 201710665651A CN 109385411 A CN109385411 A CN 109385411A
- Authority
- CN
- China
- Prior art keywords
- mannosidase
- beta
- seq
- mannose
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/2488—Mannanases
- C12N9/2491—Beta-mannosidase (3.2.1.25), i.e. mannanase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01025—Beta-mannosidase (3.2.1.25), i.e. mannanase
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of beta-Mannosidase and its applications.The sequence of the polypeptide of beta-Mannosidase of the invention are as follows: (1a) amino acid sequence as shown in SEQ ID NO:1；Or (2a) and SEQ ID NO:1 are at least 75% sequence identity and with the amino acid sequence with the beta-Mannosidase of SEQ ID NO:1 identical function.Invention additionally discloses a kind of expression vector of beta-Mannosidase and host cells.Beta-Mannosidase disclosed by the invention has preferable mannosidase enzyme activity stability in pH 3.5-8 and 30-65 DEG C of temperature.When being used for beta-Mannosidase of the invention to produce mannose, the yield of mannose can reach 7.1mg/mL, be about 6 times for not adding beta-Mannosidase of the present invention, have potential industrial application value.
Description
Technical field
The invention belongs to bioengineering fields, and in particular to a kind of beta-Mannosidase and its application.
Background technique
Beta-Mannosidase (EC3.2.1.25) belongs to a kind of excision enzyme of hemicellulose enzyme system, it can catalyzing hydrolysis Isosorbide-5-Nitrae-
β-D glycosidase cuts mannose in non-reducing end, is widely used in industries such as food, pharmacy, petroleum and bioconversions
(Mccleary B V, Nurthen E, Taravel F R, et al.Characterisat ion of
oligosaccharides produced on hydrolysis of galactomannan with β-D-m annanase
[J] .Carbohydrate Research, 1983,118 (JUL): 91-109) it can especially substitute chemical method production mannose.
Mannose dissolubility is good, is not easy to crystallize, and has potential application in food and pharmaceutical industry.Beta-Mannosidase is another
A application is to turn glucosides ability synthesis manna oligosacchride using it, produces functional manna oligosacchride to substitute chemical method
(TaubkenN, ThiemJ.Enzymatic Synthesis of Alkyl and Hydroxyalkyl β-D-
Mannopyranosides [J] .Synthesis, 1992,1992 (6): 517-518.), manna oligosacchride can not only enhance animal
Immunocompetence reduces the incidence of enterogastric diseases and the death rate of animal, and can also improve the daily gain and feed of animal
(Yue Wenbin, Che Xiangrong, subtract for army building conversion ratio, wait the influence of Mannanoligosaccharidon on Intestinal Microflora of Weaned Pigs and immunity function
[J] Journal of Shanxi Agricultural University (natural science edition), 2002,22 (2): 97-101).Therefore, to the exploitation of beta-Mannosidase
With important industrial applications prospect.
It is external oneself have a small number of microbe-derived beta-Mannosidase Gene clone and expressions report (ChauhanPS,
GuptaN.Insight into microbial mannosidases:a review [J] .Critical Reviews inB
Iotechnology, 2016,8:1-12.), but the molecular weight of most of beta-Mannosidases reported in the literature is larger, 90
~130KDa, it is difficult to express, limit the application of beta-Mannosidase.
Summary of the invention
The purpose of the present invention is to provide a kind of polypeptide of beta-Mannosidase, its encoding gene, express the β-mannose
Bacterial strain, host cell and the expression vector of the polypeptide of glycosides enzyme, and a kind of application of beta-Mannosidase is provided.
To achieve the above object, the technical solution used in the present invention are as follows: a kind of polypeptide of beta-Mannosidase, the β-
The sequence of the polypeptide of mannosidase are as follows:
(1a) amino acid sequence as shown in SEQ ID NO:1；Or
(2a) is at least 75% sequence identity and sweet with SEQ ID NO:1 β-having the same with SEQ ID NO:1
Reveal the amino acid sequence of glycosidase function.
The present invention provides the encoding gene of the polypeptide of above-mentioned beta-Mannosidase, the novel beta-Mannosidase polypeptide
Encoding gene be made of following sequence:
Nucleotide sequence shown in (1b) SEQ ID NO:2；Or
(2b) is at least 75% sequence identity and sweet with SEQ ID NO:2 β-having the same with SEQ ID NO:2
Reveal the nucleotide sequence of glycosidase function.
The encoding gene of beta-Mannosidase of the present invention derives from the horizontal mould (Lic of stalk of the racemosus voluntarily screened
Htheimia ramose), it is lower with the similitude of beta-Mannosidase gene disclosed in gene database, it is a kind of novel
5 family's beta-Mannosidase of glycosyl hydrolase, amino acid sequence is as shown in SEQ ID NO:1, and the beta-Mannosidase is by 444
A amino acid composition, relative molecular weight about 60kDa are easy to express compared with traditional beta-Mannosidase.β-of the invention is sweet
Dew glucosides enzyme polypeptide can also for containing with SEQ ID NO:1 with by BLAST algorithm measured at least 75% (such as 80%,
90%, 95% or 99%) the sequence of amino acid similarity, and the polypeptide has beta-Mannosidase activity.With the β-mannose
Highest glycosides enzyme amino acid identity is Lichtheimia corymbiferaJMRC:FSU:9682glycoside
The consistency of both hydrolase family5protein (sequence number: CDH53051.1) is 305/408 (75%)；Followed by
The consistency of both Rhizomu cor Miehei rhizomucor miehei mannosidase (sequence number: AGV01048.1) is 312/
425 (73%).
The present invention also provides a kind of expression vector, expression vector of the invention by above-mentioned beta-Mannosidase coding base
Cause and carrier is carrier composition.
Preferably, the carrier is carrier is yeast expression vector.
Preferably, the basal expression carrier is pPIC9, pPIC9k or pHIL-S1, pPICZ α A, one in pYAM75P
Kind.
It is highly preferred that the yeast expression vector is pPICZ α A.
The present invention provides a kind of beta-Mannosidase expression casette, the Expression element of the expression casette includes opening
Mover, above-mentioned encoding gene and terminator.
Preferably, the Expression element from 5 ' to 3 ' of the expression casette is followed successively by 1 promoter of AOX, promotes β-mannose
The signal peptide of glycosides enzyme secretion, 1 terminator of encoding gene and AOX of beta-Mannosidase.The wherein volume of beta-Mannosidase
Code gene order is as shown in SEQ ID NO:2.
The present invention provides a kind of genetically engineered host cell, the host cell includes expression vector or gene
Expression cassette.Preferably, the host cell is methanotrophic yeast host cells.
It is highly preferred that the host cell is pichia pastoris yeast.
The present invention provides the polypeptide, the encoding gene, the expression vector, the expression casette, the places
Chief cell is preparing the application in beta-Mannosidase.
The present invention provides a kind of application of beta-Mannosidase in terms of producing mannose.
Preferably, application of the beta-Mannosidase in terms of producing mannose is beta-Mannosidase in enzymatic hydrolysis locust tree
Bean gum produces the application in terms of mannose.
Compared with prior art, the invention has the benefit that
(1) the present invention provides a kind of beta-Mannosidase polypeptide and its encoding gene, beta-Mannosidase polypeptide tools
There is at least 75% (such as 80%, 90%, 95% or the 99%) sequence of amino acid similarity, and should measured by BLAST algorithm
Polypeptide has beta-Mannosidase activity.
(2) sweet when being used for producing using beta-Mannosidase expressed by beta-Mannosidase gene of the invention
When dew sugar, it is about 6 times for not adding beta-Mannosidase of the present invention that the yield of mannose, which can reach 7.1mg/mL, is had latent
Industrial application value.
Detailed description of the invention
Fig. 1 is pPICZ α A-LrMan5 expression vector schematic diagram；
Fig. 2 is the bacterium colony PCR figure in embodiment 2；
Fig. 3 is the SDS-PAGE figure of beta-Mannosidase LrMan5；
Fig. 4 is that pH influences result figure to LrMan5 enzyme activity；
Fig. 5 is that temperature influences result figure to LrMan5 enzyme activity；
Fig. 6 is the pH stability result figure of LrMan5；
Fig. 7 is the temperature stability result figure of LrMan5；
Fig. 8 is the kinetic constant analysis chart of LrMan5；
Fig. 9 is the HPLC figure of LrMan5 and mannosan Synergistic degradation locust bean gum production mannose.
Specific embodiment
Purpose in order to better illustrate the present invention, technical scheme and beneficial effects, below in conjunction with attached drawing and specific implementation
The invention will be further described for example.It should be noted that following the methods of implementing are explained further to what the present invention was done
It is bright, it should not be taken as limitation of the present invention.If material used in the embodiment of the present invention, reagent all can be from without specified otherwise
Commercial sources obtain.
The clone of 1 beta-Mannosidase LrMan5 gene of embodiment
The genomic DNA for extracting Lichtheimia ramose, using genomic DNA as template, design primer LrMan5-F/
R, sequence obtain beta-Mannosidase (LrMan5) as shown in table 1 (SEQ ID:3/SEQ ID:4), by the method for Standard PCR
Gene, gene order is as shown in SEQ ID NO:1.
Table 1:LrMan5 amplimer sequence
The PCR of LrMan5 gene is obtained shown in reaction system such as table 2, reaction condition is as shown in table 3.
Table 2:PCR reaction system
Table 3:PCR reaction condition
Embodiment 2 includes the yeast expression vector building and verifying of LrMan5 encoding gene
(1) LrMan5 gene and plasmid pPICZ α A distinguish double digestion
LrMan5 Gene Double endonuclease reaction system is as shown in table 4, and plasmid pPICZ α A double enzyme digestion reaction system is as shown in table 5.
Table 4:LrMan5 Gene Double endonuclease reaction system
Table 5: plasmid pPICZ α A double enzyme digestion reaction system
(2) connection of pPICZ α A carrier and LrMan5 gene
Linked system is as shown in table 6, construction of expression vector pPICZ α A-LrMan5 (expression vector map is as shown in Figure 1),
Escherichia coli DH5a is converted, the screening and amplification of carrier are carried out.
Table 6:pPICZ α A carrier and LrMan5 linked system
(3) it converts
A, pichia pastoris yeast competent cell is prepared
The well-grown Pichia pastoris single colonie of picking, is inoculated in the finger-type bottle of 5mL YPD, and 30 DEG C are incubated overnight.It takes
0.5mL overnight culture is forwarded in the 500mL shaking flask of the fresh culture containing 125mL.30 DEG C of 250rpm are cultivated to OD600=
1.3-1.5.In 4 DEG C, 1500g is centrifuged 3min and collects thallus, and thallus is resuspended with the sterile water of 125mL pre-cooling.As above centrifugation is used
Thallus is resuspended in the sterile water of 60mL pre-cooling.As above centrifugation is resuspended with the 1M sorbierite of 5mL pre-cooling.As above centrifugation, it is pre- with 0.25mL
Cold 1M sorbierite is resuspended.
B, pichia pastoris yeast host cell is converted
The recombinant DNA for taking the 100 above-mentioned bacterium solutions of μ L to linearize with 2 μ L about 1ug SacI restriction enzyme mixes, and is transferred to pre-
In cold electric revolving cup.5min is placed on ice.Eukaryocyte Transformation Parameters (the voltage 1.5kV, 200 Ω of resistance, electricity recommended by device
Hold 25 μ F, the electric shock time is 4-10msec) electric shock.It is added immediately the 1M sorbierite of 1mL pre-cooling, is transferred in the centrifuge tube of sterilizing and takes
On appropriate bacterium solution coating YPDS plate, 30 DEG C of constant-temperature incubations are until single colonie occurs after 2 days.
(4) identification of pPICZ α A-LrMan5 plasmid is recombinated
The Pichia pastoris single colonie grown on random picking YPDS plate extracts recombination pPICZ α A-LrMa n5 plasmid, with
5 ' AOX and 3 ' AOX are primer (as shown in table 7), carry out bacterium colony PCR and identify recon.Bacterium colony PCR result is as shown in Fig. 2, through surveying
Sequence proves that LrMan5 gene and carrier segments are successfully integrated on pichia pastoris X-33 genome really, in recombinant yeast really
It is real to contain corresponding foreign gene.
Bacterium colony PCR primer sequence is as shown in table 7 (SEQ ID:5/SEQ ID:6)；Bacterium colony PCR reaction system is as shown in table 8；
Bacterium colony PCR reaction condition is as shown in table 9.
Table 7: bacterium colony PCR primer sequence
Table 8: bacterium colony PCR reaction system
Table 9: bacterium colony PCR reaction condition
(5) extraction of pPICZ α A-LrMan5 plasmid is recombinated
It is as follows to recombinate pPICZ α A-LrMan5 plasmid extraction operation method:
1) bacterium solution for taking 1ml to be incubated overnight, 12000g abandon supernatant from 1min；
2) 100 μ L are added and break bacterium buffer, electric grinder grinds thallus 1-2min；
3) 500 μ L binding buffer (the combination liquid in DNA cycle pure kit) is added, is mixed by inversion；
4) 12000g is centrifuged 5min, and supernatant is taken to be added in centrifugal column, and 10000g is centrifuged 1min；
5) liquid in collecting pipe is abandoned, 500 μ L washing buffer, 10000g are added into centrifugal column and are centrifuged 1min；
6) step 5) is repeated；
7) liquid in collecting pipe is abandoned, 10000g is centrifuged 2min, centrifugal column is transferred in new 1.5mL centrifuge tube, is added
Enter 30 μ L sterile waters (65 DEG C of preheatings), 37 DEG C of placements 5min, 10000g are centrifuged 1min.Recombinant plasmid is successfully integrated into and finishes red ferment
On female X-33 genome.
The expression and purification of 3 recombinant beta mannosidase LrMan5 of embodiment
The well-grown Pichia pastoris single colonie of picking is inoculated in the finger-type bottle of the BMG containing 5mL, 30 DEG C of shake culture mistakes
Night.The bacterium solution of overnight incubation is forwarded in the 500mL triangular flask containing 50mL YPDG, is sealed with eight layers of gauze, is guaranteed good
Ventilative environment, 28 DEG C, 250rpm overnight incubation.The bacterium solution 1500g of overnight incubation is centrifuged 3min, it is fresh that 50mL is resuspended
BMMY culture is sealed, 28 DEG C, 250rpm shaken cultivation based in 500mL triangular flask with eight layers of gauze.Methanol is added extremely every 6h
The methanol of final concentration of 1.2-1.5%.Every 12h takes appropriate culture solution measurement OD600 value and enzyme activity, to determine recombinant protein
The best harvest time of expression.Tunning 6000rpm is centrifuged 10min, HITRAPQFF chromatographic column chromatography is crossed, passes through SDS-
PAGE analyzes purified product.
As a result as shown in figure 3, there is obvious band near 66KDa, it is consistent with expected size, shows that LrMan5 is finishing red ferment
Successful expression in mother.
The production method of 4 beta-Mannosidase of embodiment
(1) after level-one kind second level kind, the Pichia pastoris containing recombination pPICZ α A-LrMan5 plasmid is inoculated in BSM
In fermentation medium, culture for 24 hours, continues feed supplement glycerol, and culture to thallus OD value reaches 300.
(2) it carries out methanol induction 7 days, recombinant yeast pichia pastoris is made to produce novel beta-Mannosidase.
(3) beta-Mannosidase is separated from fermentation liquid.
The research of 5 LrMan5 enzymatic property of embodiment
LrMan5 enzyme activity determination is that substrate measures beta-Mannosidase with p-nitrophenol-β-D mannoside (p-NPM)
Vigor:
(1) the 150 μ L of buffer of pH 5.5 is taken, 50 μ Lp-NPM substrate solutions are added, is put into water-bath and preheats 5min,
The suitably diluted enzyme solution of 50 μ L, insulation reaction 10min at 65 DEG C is added.
(2) 1mL1M Na is added2CO3Solution is to terminate reaction.
(3) 200 μ L reaction solutions are drawn and enter 96 hole elisa Plates, microplate reader measures the light absorption value under 405nm.According to making before
P-NP standard curve, the amount of p-NP is generated in counting system.
Calculation formula is as follows:
In formula:
The vigor of X- sample beta-Mannosidase, U/mL；
The p-NP amount that A- is checked in from standard curve, μm oL；
N- extension rate (total extension rate)；
The enzyme solution volume that V- is added, mL；
10-10 minutes reaction time.
The relative error of two parallel determination values of same sample is no more than 8.0%, and the average value of the two is final enzyme activity
Power measured value.
The definition of enzyme activity unit (U): under most suitable determination condition, enzyme amount needed for generating the pNP of 1 μm of ol per minute is fixed
Justice is an enzyme activity unit (U), and wherein the enzyme activity of sample is indicated with U/g (solid enzyme) or U/mL (liquid enzymes).
Influence of 6 pH of embodiment to LrMan5 enzyme activity
At 50 DEG C, enzyme activity of the beta-Mannosidase under different pH (3.0~10.0) buffer conditions is measured, it is used
Buffer is one disodium hydrogen phosphate buffer of 0.2M citric acid.
It as a result is 5.5 in pH as shown in figure 4, showing beta-Mannosidase activity all with higher between 4.5-6.0
When, that is, reach highest enzyme activity, slowly declines as pH increases enzyme activity, when pH is 6.5, retain 50% enzyme activity.
Influence of 7 temperature of embodiment to LrMan5 enzyme activity
After determining optimal pH, the enzyme activity under different temperatures (35~75 DEG C) is measured under the conditions of 5.5 pH, determines enzyme
Optimum temperature.
As a result as shown in figure 5, showing that beta-Mannosidase LrMan5 reaches maximum value when temperature is 65 DEG C, when temperature is super
When crossing 65 DEG C, enzyme activity sharply declines, and enzyme activity is reduced to 40% when to 75 DEG C.
The measurement of 8 LrMan5pH tolerance of embodiment
Enzyme is stored in the buffer of different pH value, 40 DEG C save 1 hour, most suitable action pH and optimum temperature item
Enzyme activity (the one disodium hydrogen phosphate buffer of 0.2M citric acid of pH5.5,65 DEG C of reaction 10min) are measured under part.
The enzyme measures the pH stability of LrMan5 in the range of pH3-8.0, as a result as shown in fig. 6, LrMan5 is in pH
3.5-8 has preferable stability.
The measurement of 9 LrMan5 temperature tolerance of embodiment
By enzyme (30~70 DEG C) preservation 0.5h at different temperatures, enzyme activity is measured under the conditions of optimal pH and optimum temperature
(the one disodium hydrogen phosphate buffer of 0.2M citric acid of pH5.5,65 DEG C of reaction 10min).
As a result as shown in fig. 7, showing that LrMan5 has preferable thermal stability, 65 DEG C of processing 0.5h still have 80% or more
Enzyme activity.All there is 90% or more enzyme activity at 60 DEG C and following temperature processing 0.5h.
The analysis of 10 LrMan5 kinetic constant of embodiment
Under optimum condition, using pNP-M as substrate, beta-Mannosidase Rate activity is measured under different concentration of substrate.With
5 software of GraphPad Prism is analyzed.
As a result as shown in figure 8, showing the Km of the enzyme and Vmax is respectively 27.44mM and 18.65 μm of ol/mimmg.
The HPLC of 11 mannase of embodiment and beta-Mannosidase synergistic effect production mannose is analyzed
Using mannose as standard sample, locust bean gum is substrate, is separately added into mannase and mannase and β-
Mannosidase is in 50 DEG C of effect 1h.Then 10min will be boiled in reactant boiling water and terminates reaction, and room temperature is cooling, 12000rpm
It is centrifuged 20min.HPLC analysis is carried out using Shimadzu chromatograph.Chromatographic condition: chromatographic column is Alltima Aminoz nh 2 column
(4.6x250mm, 5 μm)；Mobile phase is second eyeball: water (75: 25)；Flow velocity is 1mL/min；Column temperature is 40 DEG C.
As a result as shown in figure 9, wherein each digital number is expressed as 1: 0.1g locust bean gum+90U mannase；2:
0.1g locust bean gum+180U mannase；3: 0.1g locust bean gum+360U mannase；4: 0.1g locust bean gum+
1440U mannase；5: 5mg/mL mannose standard specimen；6: 0.1g locust bean gum+720U mannase+0.012U
LrMan5；7: 0.1g locust bean gum+720U mannase+0.025U LrMan5；8: 0.1g locust bean gum+720U sweet dew is poly-
Carbohydrase+0.050U LrMan5；9: 0.1g locust bean gum+720U mannase+0.100U LrMan5；10: 0.1g locust bean
Glue+720U mannase+0.25U LrMan5.
Show that, even if additive amount reaches 14400U/g, the content of mannose is still only when only adding mannase
1.25mg/mL, and when add 7200U/g mannase while when addition 2.5U beta-Mannosidase LrMan5, mannose
Content reach 7.1mg/mL, show that the content of mannose can be effectively increased by adding beta-Mannosidase LrMan5, industrially be given birth to
Producing mannose has important application.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should
Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention
And range.
SEQUENCE LISTING
<110>Dongguan Fanyatai Biological Sci-Tech Co., Ltd.
<120>a kind of beta-Mannosidase and its application
<130> 20170803
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 444
<212> PRT
<213> Lichtheimia ramose
<400> 1
Met Val Leu Lys Thr Ser Leu Phe Thr Leu Ala Leu Ala Ile Gly Leu
1 5 10 15
Val Ser Ala Gln Pro Leu Asp Ser Arg Lys Glu His His His His His
20 25 30
Ala Asp Glu Ala Tyr Val Gln Ile Ala Lys Asp Gly Ser Gly Phe Thr
35 40 45
Arg Tyr Gly Val Pro Tyr Met Ile Arg Gly Ser Asn Tyr Trp Tyr Gly
50 55 60
Met Trp Leu Gly Ala Asp Asp Cys Asn Gly Gly Asp Arg Lys Arg Leu
65 70 75 80
Glu Thr Glu Val Arg Gln Leu Ala Glu Met Gly Val Asn Asn Leu Arg
85 90 95
Ile Met Ala Ala Ser Glu Gly Pro Asp Asp Gln Pro Tyr Arg Val Arg
100 105 110
Pro Ser Phe Met Arg Lys Pro Gly Glu Tyr Asn Glu Ala Val Phe Lys
115 120 125
Gly Leu Asp Tyr Leu Leu Asp Thr Met Asp Lys Tyr Asn Met Thr Ala
130 135 140
Val Met Thr Met Asn Asn Phe Trp His Trp Ser Gly Gly Phe Gly Gln
145 150 155 160
Tyr Val Ala Trp Ile Ser Gly Asn Gln Thr Ile Pro Tyr Pro Val Gly
165 170 175
Asp Val Thr Tyr Asp Glu Phe Thr Gln Tyr Ala Ala Arg Phe Tyr Asn
180 185 190
Asp Ser Ser Ile Ala Pro Lys Ala Gln Glu Ile Phe Lys Asn His Ile
195 200 205
Thr Thr Val Gln Asn Arg Val Asn Thr Val Asn Gly Lys Thr Tyr Lys
210 215 220
Glu Asp Pro Val Ile Met Ser Trp Gln Leu Ala Asn Glu Pro Gln Gln
225 230 235 240
Gly Pro Ala Trp Trp Phe Glu Glu Ile Ser Thr Tyr Met Lys Lys Gly
245 250 255
Ala Pro Lys His Leu Val Ser Ala Gly Leu Glu Ser Ile Leu Asp Lys
260 265 270
Tyr Asp Phe Asp Arg Ala His Lys His Lys Asn Ile Asp Tyr Thr Thr
275 280 285
Cys His Leu Trp Val Glu Asn Arg Gly Ile Tyr Asp Pro Arg Asn Val
290 295 300
Ser Thr Leu Ala Leu Ala Gln Ala Ala Ala Thr Asp Phe Ile Ser Ser
305 310 315 320
Arg Ser Glu Trp Ala Glu Thr Leu Asn Lys Pro Ile Leu Leu Glu Glu
325 330 335
Phe Gly Met Ala Arg Asp Ala Trp Arg Lys Pro Asp Asp Leu Glu Tyr
340 345 350
Glu Tyr Asn Pro Ser Thr Pro Thr Thr His Lys Asp Lys Phe Tyr His
355 360 365
Asp Ile Phe Lys Gln Ile Val Ser Leu Ala Lys Asp Gly Lys Phe Ser
370 375 380
Gly Ser Gly Phe Trp Ala Tyr Ser Gly Glu Gly Arg Ser Thr Asp Tyr
385 390 395 400
Pro Asn Lys Tyr Gly Met Val Phe Leu Gly Asp Pro Pro His Glu Pro
405 410 415
Arg Gly Trp Tyr Ser Val Tyr Asp Lys Asp Thr Thr Val Lys Val Ile
420 425 430
Arg Asp Tyr Asn Ala Glu Leu Lys Lys Leu Glu His
435 440
<210> 2
<211> 1278
<212> DNA
<213> Lichtheimia ramose
<400> 2
caaccattgg attctagaaa ggaacatcat catcatcatg ctgatgaggc ttacgttcaa 60
attgctaagg atggttcagg ttttactaga tacggtgttc cttacatgat tagaggatct 120
aactactggt acggaatgtg gttgggagct gatgattgta acggagggga cagaaagaga 180
ttggaaactg aagttagaca attggctgaa atgggagtta acaacttgag aattatggct 240
gcttccgaag gaccagatga tcaaccatac agagttagac cttcctttat gagaaagcca 300
ggtgaataca acgaagctgt ttttaagggt ttggattact tgttggatac tatggataag 360
tacaacatga ctgctgttat gactatgaac aacttttggc attggtccgg aggttttggt 420
caatacgttg cttggatttc aggtaaccaa actattccat acccagttgg cgacgttact 480
tacgatgagt tcactcaata cgctgctaga ttttacaacg attcttctat tgctccaaag 540
gctcaagaaa tttttaagaa ccatattact actgttcaaa acagagttaa cactgttaac 600
gggaaaactt acaaggaaga tccagttatt atgtcctggc aattggctaa cgaacctcaa 660
caaggacctg cttggtggtt tgaagaaatt tccacttaca tgaagaaggg cgcgccaaag 720
catttggttt ccgctggatt ggaatctatt ttggataagt acgattttga tagggcccat 780
aagcataaga acattgatta cactacttgt catttgtggg ttgaaaacag aggtatatac 840
gatcctagaa acgtttccac tttggctttg gctcaagctg ctgctactga ttttatttct 900
tctagatccg aatgggctga aactttgaac aagcctattt tgttggaaga atttggtatg 960
gctagagatg cttggagaaa gccagatgat ttggaatacg aatacaaccc atccactcca 1020
actactcata aggataagtt ttaccatgat atttttaagc aaattgtttc cttggctaag 1080
gatgggaagt tttcaggttc cggattttgg gcttactccg gtgaaggtag atccactgat 1140
taccctaaca agtacggtat ggtcttcttg ggagatcctc cacatgaacc tagaggatgg 1200
tactccgttt acgataagga tactactgtt aaggttatta gagattacaa cgctgaattg 1260
aagaagttgg aacattaa 1278
<210> 3
<211> 31
<212> DNA
<213>artificial synthesized
<400> 3
atagaattcc aaccattgga ttctagaaag g 31
<210> 4
<211> 33
<212> DNA
<213>artificial synthesized
<400> 4
atagcggccg cttaatgttc caacttcttc aat 33
<210> 5
<211> 21
<212> DNA
<213>artificial synthesized
<400> 5
gactggttcc aattgacaag c 21
<210> 6
<211> 21
<212> DNA
<213>artificial synthesized
<400> 6
gcaaatggca ttctgacatc c 21
Claims (10)
1. a kind of polypeptide of beta-Mannosidase, which is characterized in that the sequence of the polypeptide of the beta-Mannosidase are as follows:
(1a) amino acid sequence as shown in SEQ ID NO:1；Or
(2a) and SEQ ID NO:1 have at least 75% sequence identity and with SEQ ID NO:1 β-mannose having the same
The amino acid sequence of glycosides enzyme function.
2. a kind of encoding gene of beta-Mannosidase, which is characterized in that the nucleotide of the volume gene of the beta-Mannosidase
Sequence are as follows:
Nucleotide sequence shown in (1b) SEQ ID NO:2；Or
(2b) and SEQ ID NO:2 have at least 75% sequence identity and with SEQ ID NO:2 β-mannose having the same
The nucleotide sequence of glycosides enzyme function.
3. a kind of expression vector, it is characterised in that: the expression vector is by encoding gene as claimed in claim 2 and carrier is carrier
Composition；Preferably, the carrier is carrier is yeast expression vector.
4. a kind of beta-Mannosidase expression casette, it is characterised in that: the Expression element of the expression casette includes starting
Son, encoding gene and terminator as claimed in claim 2.
5. a kind of genetically engineered host cell, it is characterised in that: the host cell includes table as claimed in claim 3
Up to carrier or expression casette as claimed in claim 4.
6. host cell as claimed in claim 5, the host cell is methanotrophic yeast host cells；It is preferred that
Ground, the host cell are pichia pastoris yeast.
7. polypeptide as described in claim 1, encoding gene as claimed in claim 2, expression vector as claimed in claim 3, such as
Expression casette described in claim 4, or host cell is preparing β-mannoside as described in any one of claim 5~6
Application in enzyme.
8. a kind of beta-Mannosidase, it is characterised in that: any one in following (a)~(d):
(a) polypeptide of the beta-Mannosidase is as described in claim 1；
(b) encoding gene of the beta-Mannosidase is as claimed in claim 2；
(c) beta-Mannosidase is by expression vector as claimed in claim 3 or expression casette table as claimed in claim 4
It reaches；
(d) beta-Mannosidase is obtained as culture host cell as described in any one of claim 5~6.
9. beta-Mannosidase as claimed in claim 8 is preparing the application in mannose.
10. application as claimed in claim 9, it is characterised in that prepare mannose by hydrolyzing locust bean gum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710665651.6A CN109385411B (en) | 2017-08-04 | 2017-08-04 | Beta-mannosidase and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710665651.6A CN109385411B (en) | 2017-08-04 | 2017-08-04 | Beta-mannosidase and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109385411A true CN109385411A (en) | 2019-02-26 |
| CN109385411B CN109385411B (en) | 2021-10-26 |
Family
ID=65413584
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710665651.6A Active CN109385411B (en) | 2017-08-04 | 2017-08-04 | Beta-mannosidase and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109385411B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113337528A (en) * | 2021-06-29 | 2021-09-03 | 浙江农林大学 | Engineering strain of mannosidase and application thereof |
| CN115725546A (en) * | 2022-10-13 | 2023-03-03 | 湖南大学 | Alpha-mannosidase with function of activating plant immunity and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6566114B1 (en) * | 1998-06-10 | 2003-05-20 | Novozymes, A/S | Mannanases |
| CN101724614A (en) * | 2010-01-12 | 2010-06-09 | 中南大学 | Acid beta-mannase, genes, engineering bacteria and structure thereof |
| CN102978222A (en) * | 2012-12-19 | 2013-03-20 | 南京林业大学 | Extremely-heat-resistant beta-mannosidase gene as well as expression protein and application thereof |
| US20140331364A1 (en) * | 2011-12-19 | 2014-11-06 | Novozymes Inc. | Polypeptides Having Beta-Glucosidase Activity and Polynucleotides Encoding Same |
-
2017
- 2017-08-04 CN CN201710665651.6A patent/CN109385411B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6566114B1 (en) * | 1998-06-10 | 2003-05-20 | Novozymes, A/S | Mannanases |
| CN101724614A (en) * | 2010-01-12 | 2010-06-09 | 中南大学 | Acid beta-mannase, genes, engineering bacteria and structure thereof |
| US20140331364A1 (en) * | 2011-12-19 | 2014-11-06 | Novozymes Inc. | Polypeptides Having Beta-Glucosidase Activity and Polynucleotides Encoding Same |
| CN102978222A (en) * | 2012-12-19 | 2013-03-20 | 南京林业大学 | Extremely-heat-resistant beta-mannosidase gene as well as expression protein and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| CDS12333.1: "hypothetical protein LRAMOSA04528 [Lichtheimia ramosa]", 《GENBANK》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113337528A (en) * | 2021-06-29 | 2021-09-03 | 浙江农林大学 | Engineering strain of mannosidase and application thereof |
| CN115725546A (en) * | 2022-10-13 | 2023-03-03 | 湖南大学 | Alpha-mannosidase with function of activating plant immunity and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109385411B (en) | 2021-10-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Deng et al. | Heterologous expression and characterization of an antifungal chitinase (Chit46) from Trichoderma harzianum GIM 3.442 and its application in colloidal chitin conversion | |
| Xu et al. | β-1, 3-1, 4-glucanase gene from Bacillus velezensis ZJ20 exerts antifungal effect on plant pathogenic fungi | |
| CN101492661B (en) | Clone, expression of beta-glucosidase gene, and preparation for gentian oligose | |
| CN101457207B (en) | Eosinophil beta-mannanase MAN5A and gene and application thereof | |
| CN105821020B (en) | A kind of 'beta '-mannase mRmMan5A and its encoding gene and application | |
| CN101724614A (en) | Acid beta-mannase, genes, engineering bacteria and structure thereof | |
| Su et al. | Extracellular expression of a novel β-agarase from Microbulbifer sp. Q7, isolated from the gut of sea cucumber | |
| CN105624137A (en) | Sodium alginate lyase Algb and its coding gene and application thereof | |
| CN102676477A (en) | Transformation of acidic beta-mannase gene and construction of engineering bacteria of acidic beta-mannase gene | |
| CN110054702A (en) | Zearalenone degradation enzyme fusion proteins and its encoding gene and application | |
| Song et al. | Isolation and characterization of a novel glycosyl hydrolase family 74 (GH74) cellulase from the black goat rumen metagenomic library | |
| CN110527677A (en) | Zearalenone hydrolyzes enzyme mutant ZHDM2 and its encoding gene and application | |
| CN109385411A (en) | A kind of beta-Mannosidase and its application | |
| CN103421707B (en) | Tibetan pig source bacillus and application thereof | |
| CN109957520A (en) | Exogenous gene expression Pichi strain | |
| CN109679938A (en) | A kind of chitinase Chit46 and its expression and purification method and application | |
| CN102363774B (en) | Beta-mannaseBA-Man5A with wide pH range, gene thereof and application of gene | |
| CN100420744C (en) | Beta-mannase, its expression and special engineering bacterium | |
| CN114350641B (en) | Ruminant rumen protozoan xylanase OGXyn1A and application thereof | |
| CN101921739B (en) | Low-temperature xylanase XYNGR40 and gene and application thereof | |
| JP2007061038A (en) | Chitinase derived from bacterium of genus paenibacillus and gene encoding the same | |
| CN110358780A (en) | A kind of gene encoding 'beta '-mannase, and the recombinant plasmid containing the gene and recombinant bacterium and its construction method | |
| CN102978187A (en) | High-activity beta-mannanase MAN5A with pH value within range of 2.5-6.5, and gene and application thereof | |
| CN103602646B (en) | The beta-glucoside enzyme mutant that a kind of optimal reactive temperature improves and application thereof | |
| Prakash et al. | Production and partial purification of protease by selected bacterial strains using raw milk as substrate |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |